Inflammation is normally an acute response by the immune system to invasion by microbial pathogens, chemicals or physical injury. In some cases, however, the inflammatory response can progress to a chronic state, and be the cause of inflammatory disease. Therapeutic control of this chronic inflammation in diverse diseases is a major medical need.
Leukotrienes (LT) are biologically active metabolites of arachidonic acid (B. Samuelsson, Science 1983, 220(4597): 568-575) that have been implicated in inflammatory diseases, including asthma (D. A. Munafo et al., J. Clin. Invest. 1994, 93(3):1042-1050; N. Miyahara, et al., Allergol Int., 2006, 55(2): 91-7; E. W. Gelfand, et al., J. Allergy Clin. Immunol. 2006, 117(3): 577-82; K. Terawaki, et al., J. Immunol. 2005, 175(7): 4217-25), inflammatory bowel disease (IBD) (P. Sharon and W. F. Stenson, Gastroenterology 1984, 86(3): 453-460), chronic obstructive pulmonary disease (COPD) (P. J. Barnes, Respiration 2001, 68(5): 441-448), arthritis (R. J. Griffiths et al., Proc. Natl. Acad. Sci. U.S.A. 1995, 92(2): 517-521; F. Tsuji et al., Life Sci. 1998, 64(3): L51-L56), psoriasis (K. Ikai, J. Dermatol. Sci. 1999, 21(3): 135-146; Y. I. Zhu and M. J. Stiller, Skin Pharmacol. Appl. Skin Physiol. 2000, 13(5):235-245) and atherosclerosis (Friedrich, E. B. et al. Arterioscler Thromb Vasc Biol 23, 1761-7 (2003); Subbarao, K. et al. Arterioscler Thromb Vasc Biol 24, 369-75 (2004); Helgadottir, A. et al. Nat Genet. 36, 233-9 (2004); Jala, V. R. et al Trends in Immun. 25, 315-322 (2004)). The synthesis of leukotrienes is initiated by the conversion of arachidonic acid to an unstable epoxide intermediate, leukotriene A4 (LTA4), by 5-lipoxygenase (5-LO) (A. W. Ford-Hutchinson et al., Annu. Rev. Biochem. 1994, 63: 383-347). This enzyme is expressed predominantly by cells of myeloid origin, particularly neutrophils, eosinophils, monocytes/macrophages and mast cells (G. K. Reid et al., J. Biol. Chem. 1990, 265(32): 19818-19823). LTA4 can either be conjugated with glutathione by leukotriene C4 (LTC4) synthase to produce the cysteinyl leukotriene, LTC4, or hydrolyzed to the diol, leukotriene B4 (LTB4) (B. Samuelsson, Science 1983, 220(4597): 568-575). LTC4 and its metabolites, LTD4 and LTE4, induce smooth muscle contraction, broncho-constriction and vascular permeability, while LTB4 is a potent chemo-attractant and activator of neutrophils.
The stereospecific hydrolysis of LTA4 to LTB4 is catalyzed by leukotriene A4 hydrolase (LTA4H), a zinc-containing, cytosolic enzyme. This enzyme is ubiquitously expressed, with high levels in small intestinal epithelial cells, lung, and aorta (B. Samuelsson and C. D. Funk, J. Biol. Chem. 1989, 264(33): 19469-19472). Moderate expression of LTA4H is observed in leukocytes, particularly neutrophils (T. Yokomizo et al., J. Lipid Mediators Cell Signalling 1995, 12(2,3): 321-332).
Leukotriene B4 is a key pro-inflammatory mediator, able to recruit inflammatory cells, such as neutrophils and eosinophils, as well as activate neutrophils (F. A. Fitzpatrick et al., Ann. N.Y. Acad. Sci. 1994, 714: 64-74; S. W. Crooks and R. A. Stockley, Int. J. Biochem. Cell Biol. 1998, 30(2): 173-178; A. Klein et al., J. Immunol. 2000, 164: 4271-4276). LTB4 mediates its pro-inflammatory effects by binding to G protein-coupled receptors, leukotriene B4 receptor 1 (BLT1) and leukotriene B4 receptor 2 (BLT2) (T. Yokomizo et al., Arch. Biochem. Biophys. 2001, 385(2): 231-241). The receptor first identified, BLT1, binds LTB4 with high affinity, leading to intracellular signaling and chemotaxis. BLT1 is expressed mainly in peripheral leukocytes, particularly neutrophils, eosinophils, macrophages (Huang, W. W. et al. J Exp Med 188, 1063-74 (1998)) and monocytes (Yokomizo, T., Izumi, T. & Shimizu, T. Life Sci 68, 2207-12 (2001)). The murine receptor is also expressed on effector T cells and was recently shown to mediate LTB4-dependent migration of effector CD8+ T cells (Goodarzi, K., Goodarzi, M., Tager, A. M., Luster, A. D. & von Andrian, U. H. Nat Immunol 4, 965-73 (2003); Ott, V. L., Gambier, J. C., Kappler, J., Marrack, P. & Swanson, B. J. Nat Immunol 4, 974-81 (2003)), early effector CD4+ T helper type 1 (TH1) and TH2 chemotaxis and adhesion to endothelial cells, as well as early effector CD4+ and CD8+ T cell recruitment in an asthma animal model (Tager, A. M. et al., Nat Immunol 4, 982-90 (2003)). LTB4 receptor BLT2 (S. Wang et al., J. Biol. Chem. 2000, 275(52): 40686-40694; T. Yokomizo et al., J. Exp. Med. 2000, 192(3): 421-431) shares 42% amino acid homology with BLT1, but is more broadly expressed, including in peripheral tissues such as the spleen, ovary and liver, as well as in leukocytes. BLT2 binds LTB4 with lower affinity than BLT1 does, mediates chemotaxis at higher concentrations of LTB4, and differs from BLT1 in its affinity for certain antagonists. While LTB4 receptor antagonists may differ in their affinity for BLT1 versus BLT2, blocking the production of LTB4 using LTA4H inhibitors would be expected to inhibit the downstream events mediated through both BLT1 and BLT2.
Studies have shown that introduction of exogenous LTB4 into normal tissues can induce inflammatory symptoms (R. D. R. Camp et al., Br. J. Pharmacol. 1983, 80(3): 497-502; R. Camp et al., J. Invest. Dermatol. 1984, 82(2): 202-204). Elevated levels of LTB4 have been observed in a number of inflammatory diseases including IBD, COPD, psoriasis, rheumatoid arthritis (RA), cystic fibrosis and asthma (S. W. Crooks and R. A. Stockley, Int. J. Biochem. Cell Biol. 1998, 30(2): 173-178). Therefore, reduction of LTB4 production by an inhibitor of LTA4H activity would be predicted to have therapeutic potential in a wide range of diseases.
This idea is supported by a study of LTA4H-deficient mice that, while otherwise healthy, exhibited markedly decreased neutrophil influx in arachidonic acid-induced ear inflammation and zymosan-induced peritonitis models (R. S. Byrum et al., J. Immunol. 1999, 163(12): 6810-6819). LTA4H inhibitors have been shown to be effective anti-inflammatory agents in pre-clinical studies. For example, oral administration of LTA4H inhibitor SC57461 caused inhibition of ionophore-induced LTB4 production in mouse blood ex vivo, and in rat peritoneum in vivo (J. K. Kachur et al., J. Pharm. Exp. Ther. 2002, 300(2), 583-587). Eight weeks of treatment with the same inhibitor compound significantly improved colitis symptoms in cotton top tamarins (T. D. Penning, Curr. Pharm. Des. 2001, 7(3): 163-179). The spontaneous colitis that develops in these animals is very similar to human IBD. The results therefore indicate that LTA4H inhibitors would have therapeutic utility in this and other human inflammatory diseases.
Events that elicit the inflammatory response include the formation of the pro-inflammatory mediator leukotriene B4. Hydrolase LTA4H catalyzes the formation of this mediator, and LTA4H inhibitors block the production of the pro-inflammatory mediator LTB4, thus providing the ability to prevent and/or treat leukotriene-mediated conditions, such as inflammation. The inflammatory response is characterized by pain, increased temperature, redness, swelling, or reduced function, or by a combination of two or more of these symptoms. Regarding the onset and evolution of inflammation, inflammatory diseases or inflammation-mediated diseases or conditions include, but are not limited to, acute inflammation, allergic inflammation, and chronic inflammation.
Atopic dermatitis (AD) is a chronic inflammatory skin disease that usually occurs in individuals with a personal or family history of atopy. The major features are pruritus and chronic or relapsing eczematous lesions. Complications include bacterial, fungal and viral infections as well as ocular disease. Atopic dermatitis is the most common inflammatory skin disease in children and affects more than 15% of children in the US (Laughter, D., et al., J. Am. Acad. Dermatol. 2000, 43, 649-655). Atopic dermatitis may persist in 60% of adults who were affected as children (Sidbury, R., et al., Dermatol. Clin. 2000, 18(1), 1-11).
Atopic dermatitis has significant societal impact. The family stress related to caring for children with moderate to severe AD may be comparable to the stress seen in families of children with type I diabetes mellitus (Su, J. C., et al., Arch. Dis. Child 1997, 76, 159-162). In the US, the annual cost of medical services and prescription drugs for the treatment of AD/eczema is similar to those for emphysema, psoriasis and epilepsy (Ellis, C. N., et al., J. Am. Acad. Dermatol. 2002, 46, 361-370).
Topical corticosteroids and emollients are the standard of care in the treatment of AD. However, topical steroids are associated with cutaneous complications such as striae, atrophy and telangeictasia that limit the long-term use of these agents (Hanifin, J. M., et al., J. Am. Acad. Dermatol. 2004, 50, 391-404). Emollients have a steroid-sparing effect and are useful for both prevention and maintenance therapy. Crude coal tar and preparations containing coal tar derivatives have also been used for many years in the treatment of AD and have significant cosmetic disadvantages that influence compliance (Hanifin, et al., 2004). Topical doxepin may be a useful short-term adjunctive therapy for the relief of pruritus but sedation and contact dermatitis may complicate its use (Hanifin, et al., 2004).
The topical calcineurin inhibitors tacrolimus (Protopic®) and pimecrolimus (Elidel®) have been shown to reduce the extent, severity and symptoms of AD in adults and children and are approved for use as second-line therapy of AD. However, the recent addition of boxed warnings to the product labels regarding rare cases of malignancy reported in patients treated with topical calcineurin inhibitors limits long term use of these agents in the treatment of AD (Food and Drug Administration [FDA]/Center for Drug Evaluation and Research [CDER] resources page).
Antibiotics are used in the treatment of Staphylococcus aureus infections in patients with AD but have a minimal effect on the dermatitis (Hanifin, et al., 2004). Although sedating antihistamines may be useful if sleep disruption is present, oral antihistamines are generally not effective in treating AD-associated pruritus (Hanifin, et al., 2004). Ultraviolet (UV) phototherapy, including photochemotherapy with psoralen is well established in the treatment of AD but relapse upon cessation of therapy frequently occurs (Hanifin, et al., 2004).
Systemic immunomodulatory therapy with cyclosporine and corticosteroids is effective but can be associated with severe side effects and is generally reserved for patients with severe disease. Systemic corticosteroids are associated with growth retardation in children, avascular necrosis of bone, osteopenia, increased risk of infection, poor wound healing, cataracts, hyperglycemia and hypertension. Cyclosporine is nephrotoxic in a majority of patients and is associated with tremor, hirsutism, hypertension, hyperlipidemia and gum hyperplasia.
While AD that is mild to moderate in severity generally responds to topical therapy, correct use of these therapies and compliance remain a major issue in the clinic. An oral or topical agent lacking the risks associated with corticosteroids and the calcineurin inhibitors would be a welcome addition to the armamentarium of treatments for AD that is mild to moderate in severity. An effective oral or topical therapy with fewer side effects than systemic immunomodulatory therapies and potent topical corticosteroids would fill an unmet medical need in the treatment of AD.
LTB4 is a potent pro-inflammatory lipid mediator derived from arachidonic acid via the 5-lipoxygenase (5-LO) pathway. LTB4 is known to be a chemotactic factor and activator of leukocytes, particularly granulocytes and T-cells, and has been implicated in several allergic and inflammatory diseases.
LTB4 plays a role in AD. LTB4 levels are elevated in skin lesions and plasma in AD. Reported in vivo and in vitro studies have shown that leukotrienes, especially LTB4, contribute to the inflammation of the skin in AD through their chemotactic effect on inflammatory cells. LTB4 receptors are expressed on mast cells, T cells, eosinophils, dendritic cells and macrophages, all of which accumulate in AD lesions. LTB4 itself is a pruritic agent, and has also been shown to mediate substance P- and nociceptin-induced pruritus, a key component of the itching in AD. LTB4 also induces proliferation of keratinocytes, an effect which is further potentiated by substance P. Recent reports indicate a role for LTB4 in development of a Th2 immune response and IgE production. The role of LTB4 in AD is further supported by beneficial effects of the 5-lipoxygenase inhibitor, zileuton, in a small open-label trial in AD (Woodmansee, D. P., et al., Ann. Allergy Asthma Immunol. 1999, 83, 548-552) and in relieving the pruritus in Sjögren-Larsson syndrome patients who have elevated LTB4 levels due to an impairment in its degradation (Willemsen, M. A., et al., Eur. J. Pediatr. 2001, 160, 711-717).
Embodiments of this invention have shown dose-dependent inhibition of dermal inflammation and pruritus in a number of preclinical models, as well as inhibition of Th2 responses and IgE production. Oral administration of embodiments of this invention inhibited arachidonic-acid-induced ear inflammation (neutrophil influx and edema) in mice. In a mouse model of cutaneous contact hypersensitivity (CHS), dosing of embodiments of this invention around sensitization decreased IgE production and skin edema upon antigen challenge, while dosing prior to challenge decreased pruritus. Oral dosing of embodiments of this invention was also efficacious in reducing pruritus in mice induced by compound 48/80, substance P or IgE-antigen interaction in the skin.
LTA4H inhibitors are hypothesized to specifically block the production of LTB4 from LTA4, without affecting the biosynthesis of lipoxins, which are also produced from LTA4. Increasing or maintaining lipoxin A4 (LXA4) production may have beneficial therapeutic effects in dermal inflammation as it has been reported that topical application of a stable lipoxin analogue inhibits edema, granulocyte infiltration and epidermal hyperproliferation in murine skin inflammation models. 5-LO inhibitors block the pathway upstream of LTA4. This would be expected to lead to a block in not only synthesis of LTA4, LTB4 and cysteinyl leukotrienes, but also LXA4.
Embodiments of this invention have been studied in a number of in vivo skin (and peritoneal) inflammation models including arachidonic acid-induced ear inflammation, zymosan-induced peritonitis, fluorescein isothiocyanate (FITC)-induced cutaneous contact hypersensitivity (CHS), and cutaneous itch induced by substance P, compound 48/80 and IgE/antigen interaction. Pharmacology models were also performed with embodiments of this invention to assess their effects on the development of Th2 immune responses and allergic lung inflammation, including ovalbumin (OVA) sensitization model and OVA sensitization and airway challenge models. Additional pharmacological profiling demonstrated efficacy in models of acute and chronic TNBS-induced colitis and collagen-induced arthritis.
An allergy is an abnormal reaction to an allergen (an ordinarily harmless substance) that triggers an abnormal response in a sensitized individual. Allergic rhinitis is an inflammation of the mucus membranes of the nose that occurs in response to an airborne antigen (allergen). Allergic rhinitis, also called allergic rhinoconjunctivitis, is characterized by frequent or repetitive sneezing, runny or congested nose, and pruritus of the nose, eyes and throat. It may also be associated with other symptoms such as headache, impaired smell, postnasal drip, conjunctival symptoms (e.g., itchy watery eyes), sinusitis and other complicating respiratory symptoms. Depending upon the time of exposure, allergic rhinitis can be classified as perennial, seasonal or occupational.
Embodiments of this invention have shown dose-dependent inhibition of lung inflammation in a number of pre-clinical models, as well as inhibition of Th2 responses and IgE production. In addition, embodiments of this invention inhibit pruritus induced by allergen/IgE interaction.
Based upon the well-described leukotriene biosynthesis pathway (FIG. 1), LTA4H inhibitors are hypothesized to specifically block the production of LTB4 from LTA4, without affecting the biosynthesis of lipoxins, which are also produced from LTA4. Lipoxins, such as LXA4, have been the focus of intense study and are known to play a key role as natural anti-inflammatory agents and key mediators of the natural process of resolving an inflammatory response. Furthermore, production of endogenous LXA4 has been described in a variety of inflammatory diseases and lower levels of LXA4 have been found in patients with severe versus moderate asthma. These data are consistent with the proposition that LXA4 plays an important role in resolution of acute inflammation. Unlike LTA4 inhibitors, 5-LO inhibitors block this pathway upstream of LTA4. This would lead to a block in not only synthesis of LTA4, LTB4 and cysteinyl leukotrienes, but also LXA4. Furthermore, there is a possibility that LTA4H inhibitors result in a buildup of LTA4, and pathway shunting to pro-inflammatory cysteinyl leukotrienes, although to date there is no known data to support this possibility. Embodiments of this invention have shown in a model of zymosan-induced peritonitis that inhibition of LTB4 production leads to an increase in LXA4 production.
Neutrophil infiltration is a prominent feature of severe asthma. Zileuton (Zyflo®) has been suggested to be efficacious is severe asthma patients, while CysLT antagonists (i.e., Montelukast/Singulair®) are not. Embodiments of this invention inhibit Th2 T cell responses and IgE production in animal models of asthma.
Embodiments of this invention inhibited sensitization to antigen and reduced inflammatory responses to airway allergen challenge in sensitized mice, leading to dose-dependent decreases in airway hyperreactivity, airway recruitment of inflammatory cells, and reductions in interleukin (IL)-5, IL-13, and antigen-specific IgE production.
In trinitrobenzene sulfonic acid (TNBS)-induced colitis in rats, embodiments of this invention had significant inhibitory effects on colonic inflammation, including macroscopic colonic injury, inflammatory cell content, and levels of tumor necrosis factor alpha (TNF-α), LTB4, and IL-6. LTA4H inhibition by embodiments of this invention also significantly attenuated the joint inflammation and swelling associated with the destruction of collagen in murine models of arthritis.
Embodiments of this invention are expected to find utility in treating skin burns, such as those due to sunburn or some other agent.
Embodiments of this invention are expected to find utility in treating also any one or a combination of atopic dermatitis, contact dermatitis, acne (T. Alestas, et al., J. Mol. Med. 2006, 84(1): 75-87; Ch. C. Zouboulis, et al., Dermatology, 2005, 210(1): 36-8; Arch. Dermatol. 2003, 139(5): 668-70), myocardial infarction (A. Helgadottir, et al., Nat. Genet. 2006, 38(1): 68-74; Nat. Genet. 2004, 36(3): 233-9; H. Hakonarson, et al., JAMA 2005, 293(18): 2245-56), stroke (A. Helgadottir, et al., Nat. Genet. 2004, 36(3): 233-9; F. C. Barone, et al., Mol. Chem. Neuropathol. 1995, 24(1): 13-30), pain (J. M. Cunha, et al., Br. J. Pharmacol. 2003, 139(6): 1135-45; S. W. Hwang, et al., Proc. Natl. Acad. Sci. USA 2000, 97(11): 6155-60), itch (T. Andoh, et al., Eur. J. Pharmacol. 2006, 547(1-3): 59-64, 2000, 406(1): 149-152, 1998, 353(1): 93-96); J. Investigativ. Dermatol. 2004, 123(1): 196-201, 2001, 117(6): 1621-26; gingivitis (G. Emingil, et al., J. Periodontol. 2001, 72(8): 1025-31), uveitis (T. Liao, et al., Invest. Ophthalmol. Vis. Sci. 2006, 47(4): 1543-9), bronchitis (S. Gompertz, et al., Eur. Respir. J. 2001, 17(6): 1112-9), allergic rhinitis, cystic fibrosis (G. E. Carpagnano, et al., Am. J. Respir. Crit. Care Med. 2003, 167(8): 1109-12), upper gastrointestinal cancer (X. Chen, et al., Curr. Cancer Drug Targets 2004, 4(3): 267-83; J. natl. cancer inst. 2003, 95(14): 1053-61), and sepsis (H. Nakae, et al., Res. Commun. Chem. Pathol. Pharmacol. 1994, 83(2): 151-6, 84(3): 271-81), and skin burns.
Examples of textbooks on the subject of inflammation include: 1) Gallin, J. I.; Snyderman, R., Inflammation: Basic Principles and Clinical Correlates, 3rd ed.; Lippincott Williams & Wilkins: Philadelphia, 1999; 2) Stvrtinova, V., et al., Inflammation and Fever. Pathophysiology Principles of Diseases (Textbook for Medical Students); Academic Press: New York, 1995; 3) Cecil; et al. Textbook Of Medicine, 18th ed.; W.B. Saunders Co., 1988; and 4) Stedman's Medical Dictionary.
Background and review material on inflammation and conditions related with inflammation can be found in articles such as the following: C. Nathan, Points of control in inflammation, Nature 2002, 420: 846-852; K. J. Tracey, The inflammatory reflex, Nature 2002, 420: 853-859; L. M. Coussens and Z. Werb, Inflammation and cancer, Nature 2002, 420: 860-867; P. Libby, Inflammation in atherosclerosis, Nature 2002, 420: 868-874; C. Benoist and D. Mathis, Mast cells in autoimmune disease, Nature 2002, 420: 875-878; H. L. Weiner and D. J. Selkoe, Inflammation and therapeutic vaccination in CNS diseases, Nature 2002, 420: 879-884; J. Cohen, The immunopathogenesis of sepsis, Nature 2002, 420: 885-891; D. Steinberg, Atherogenesis in perspective: Hypercholesterolemia and inflammation as partners in crime, Nature Medicine 2002, 8(11): 1211-1217.
Inflammation is due to or associated with any one of a plurality of conditions, such as asthma, chronic obstructed pulmonary disease (COPD), atherosclerosis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases (including Crohn's disease and ulcerative colitis), psoriasis, atopic dermatitis, contact dermatitis, acne, myocardial infarction, stroke, pain, itch (pruritus), gingivitis, uveitis, bronchitis, allergic rhinitis, cystic fibrosis, upper gastrointestinal cancer, sepsis, and skin burns, which are each characterized by excessive or prolonged inflammation at some stage of the disease.
Aryl-substituted bridged or fused diamines are disclosed in U.S. Patent Appl. Publ. Nos. US2003/004191, US2005/043355, and US2006/074121 and in U.S. Pat. Nos. 6,559,140, 5,700,816, 5,585,492, 5,719,306, 6,506,876, 5,723,492, and 6,407,140. Benzothiazole and benzoxazole LTA4H modulators have been described in U.S. Patent Appl. Publ. Nos. US2005/0043378 and US2005/0043379. In addition, diamine derivatives are described as LTA4H inhibitors in U.S. Patent Appl. Publ. Nos. 2007/0155726 and 2007/079078. However, there remains a need for potent LTA4H modulators with desirable pharmaceutical properties. Certain aryl-substituted bridged or fused diamine derivatives have been found in the context of this invention to have LTA4H-modulating activity.